Expression Of CD86 Research Articles

Extrafollicular CD19lowCXCR5−CD11c− double negative 3 (DN3) B cells are significantly associated with disease activity in females with systemic lupus erythematosus

ObjectiveB cells play a major role in the development and maintenance of systemic lupus erythematosus (SLE). Double negative (DN) B cells defined by the lack of surface expression of IgD and CD27 have attracted recent interest for their sensitivity to Toll-like receptor 7 (TLR7) ligands and their potential role in the production of autoantibodies. Here we aimed at investigating the possible association of DN B cells and their subsets with SLE disease activity specifically in female patients, in which TLR7 gene has been reported to escape X chromosome inactivation. MethodsPeripheral blood mononuclear cells were purified from woman participating to the clinically well-characterized Swiss SLE Cohort Study (SSCS). PBMC from age-matched healthy females were used as controls. PBMC were stained for cell surface markers, intracellular Tbet and analyzed by multicolor cytofluorimetry. Single nucleotide TLR7 polymorphisms were assessed by polymerase chain reaction. ResultsThe median SLE disease activity index of the 86 females was 2, IQR [0–6], all but 8 were under chronic SLE treatment. B cells co-expressing CD11c and Tbet were increased, the mean fluorescence intensity (MFI) of CD19 was considerably reduced and we observed a large increase in CD11c + CXCR5-and CD11c-CXCR5-concomitantly with a reduction of CD11c-CXCR5+ B cells in SLE compared to 40 healthy donors (HD). When focusing on the DN B cell subset, we found a reduction of DN1 (CD11c-CXCR5+) and an increase of DN2 (CD11c + CXCR5-) and most impressively of DN3 (CD11c-CXCR5-) cells. The DN subset, particularly DN3, showed the lowest level of CD19 expression. Both DN1 and DN3 percentages as well as the CD19 MFI of DN cells were associated with SLE disease activity. The use of glucocorticoids, immunosuppressants, and antimalarials impacted differentially on the frequencies of DN B cell subsets. CD19 MFI in B cells and the percentage of DN3 were the strongest biomarkers of disease activity. The TLR7 snp3858384 G allele was associated with increased percentages of B cells and CD19+CD11c-CXCR5+ and decreased CD19+CD11c-CXCR5-. ConclusionsDN3 B cells are strongly associated with SLE clinical activity pointing to their potential involvement in disease pathogenesis, and CD19 expression level performs accurately as disease activity biomarker.

An altered natural killer cell immunophenotype characterizes clinically severe pediatric RSV infection.

Respiratory syncytial virus (RSV) infects nearly all children by 2 years of age and is a leading cause of pediatric hospitalizations. A subset of children with RSV infection (RSV+ children) develop respiratory failure requiring intensive care, but immune mechanisms distinguishing severe pediatric RSV infection are not fully elucidated. Natural killer (NK) cells are key innate immune effectors of viral host defense. In this study of 47 critically ill RSV+ children, we coupled NK cell immunophenotype and cytotoxic function with clinical parameters to identify an NK cell immune signature of severe pediatric RSV disease. Airway NK cells were increased in intubated RSV+ children with severe hypoxemia and prolonged duration of mechanical ventilation and were correlated with clinical severity scores. Peripheral blood NK cells were decreased in RSV+ patients and had altered activating receptor expression, with increased expression of CD69 and decreased expression of NKG2D. Ex vivo, circulating NK cells from RSV+ patients exhibited functional impairment characterized by decreased cytotoxicity as well as aberrant immune synapse assembly and lytic granule trafficking. NK cell frequency and phenotype correlated with clinical measures that defined disease severity. These findings implicate a role for NK cells in mediating RSV immunopathology and suggest that an altered NK cell immunophenotype is associated with severe RSV disease in young children.

Magnetically Driven Hydrogel Surfaces for Modulating Macrophage Behavior.

During the host response toward implanted biomaterials, macrophages can shift phenotypes rapidly upon changes in their microenvironment within the host tissue. Exploration of this phenomenon can benefit significantly from the development of adequate tools. Creating cell microenvironment alterations on classical hydrogel substrates presents challenges, particularly when integrating them with cell cultivation and monitoring processes. However, having the capability to dynamically manipulate the cell microenvironment on biomaterial surfaces holds significant potential. We introduce magnetically actuated hydrogels (MadSurface) tailored to induce reversible stiffness changes on polyacrylamide hydrogel substrates with embedded magnetic microparticles in a time-controllable manner. Our investigation focused on exploring the potential of magnetic fields and MadSurfaces in dynamically modulating macrophage behavior in a programmable manner. We achieved a consistent modulation by subjecting the MadSurface to a pulsed magnetic field with a frequency of 0.1 Hz and a magnetic field flux density of 50 mT and analyzed exposed cells using flow cytometry and ELISA. At the single-cell level, we identified a subpopulation for which the dynamic stiffness conditions in conjunction with the pulsed magnetic field increased the expression of CD206 in M1-activated THP-1 cells, indicating a consistent shift toward the M2 anti-inflammatory phenotype on MadSurface. At the population level, this effect was mostly hindered in the culture period utilized in this work. The MadSurface approach advances our understanding of the interplay between magnetic field, cell microenvironment alterations, and macrophage behavior.

FOXO1-mediated autophagy regulation by miR-223 in sepsis-induced immunosuppression.

Immunosuppression is the main cause of the high mortality rate in patients with sepsis. The decrease in the number and dysfunction of CD4+ T lymphocytes is crucial to the immunosuppressed state of sepsis, in turn affecting the development and prognosis of sepsis. Autophagy has been shown to play an important role in the immune imbalance exhibited during sepsis. In this study, we modulate the expression of miR-223 in CD4+ T lymphocytes, via the transfection of a mimic or an inhibitor of miR-223 to establish cell models of miR-223 overexpression and knockdown, respectively. Levels of autophagy were monitored using a double-labeled lentivirus (mRFP-GFP-LC3) and electron microscopy, and western blot analysis was used to estimate the levels of autophagy-related proteins and FOXO1 in the two cell models after co-treatment with lipopolysaccharide (LPS) and siRNA against FOXO1. We found that when the expression of miR-223 increased, FOXO1 expression decreased and autophagy decreased; whereas, when FOXO1 expression was inhibited, autophagy decreased significantly in different cell models after LPS induction. Thus, this study proved that miR-223 participate in the regulation of LPS-induced autophagy via the regulation of FOXO1 expression in CD4+ T lymphocytes which shed a new light for the diagnosis and treatment of sepsis.

Exploring the complex immunomodulatory effects and gut defense via oral administration of Astragali radix water extract to normal mice

BackgroundAstragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. It exhibits diverse biological activities, including immunomodulatory and anti-inflammatory properties; however, some of its activities have only been demonstrated in vitro.ObjectiveTo examine the effects of orally administered AR extract on immune cells and the intestine under physiological conditions, which bridges the gap between previously observed in vitro outcomes and in vivo results.MethodsAR extract was prepared by hot water extraction. Three separate animal experiments were conducted to isolate macrophages, splenocytes, and the small intestine epithelium. For the macrophage preparation experiment, an intraperitoneal injection of sterile thioglycolate was administered. The mice received oral AR extract at doses of 0.1, 0.5, or 2.5 g/kg for ten days. At the end of each experiment, cells or tissues were isolated. A portion of macrophages and splenocytes were analyzed for the phenotypic changes. The remaining cells were cultured and stimulated with lipopolysaccharide (LPS) or mitogen ex vivo to assess activation status, proliferation, and cytokine production. Samples of the intestine were subjected to real-time RT-PCR.ResultsPeritoneal macrophages from AR-treated mice exhibited increased expression of scavenger receptors, including SRA and CD36. Stimulation of these macrophages ex vivo with LPS selectively modulated the inflammatory response, including reduced expression of the costimulatory molecules CD40 and CD86, which are important for T cell responses, without affecting TNF-α and IL-6 production. Splenocytes from AR-treated mice exhibited a dose-dependent increase in CD4 and CD8 T cells; however, stimulation with mitogen decreased T cell proliferation and reduced IFN-γ production, which is essential for macrophage activation. An analysis of the small intestinal epithelium revealed an attenuated antimicrobial response, including reduced IgA content in the lumen and decreased expression of mucin-2 and polymeric Ig receptor genes.ConclusionThe response of immune cells following oral treatment with AR extract did not replicate the previously documented in vitro findings. Immune cells and intestinal epithelium from mice administered oral AR extract exhibited a selective anti-inflammatory phenotype. The overall findings indicate that the systemic effects after oral administration of AR extract include reduced sensitivity to inflammatory insults.

Prenylated Dihydroflavonol from Sophora flavescens Regulate the Polarization and Phagocytosis of Macrophages In Vitro

As an important member of innate immunity, macrophages show remarkable plasticity and heterogeneity, and play an important role in immune regulation, tissue development, homeostasis of the internal environment and injury repair. However, the excessive activation of macrophages is closely related to the occurrence and development of many diseases. The prenylated flavonoid structure is one of the characteristic structures isolated from Sophora flavescens, with anti-inflammatory, anti-tumor, anti-allergy and other effects. In this study, the effects of (2R)-3β,7,4′-trihydroxy-5-methoxy-8-prenylflavanone (TMP), a prenylated dihydroflavonol, on the polarization and phagocytosis of macrophages were systematically studied. In LPS-induced M1-type macrophages, TMP dose-dependently inhibited the expression of COX-2, iNOS and the secretion of NO, IL-1β, IL-6 and IL-18, showing an inhibitory effect on M1 polarization. Further experiments revealed that it was related to the inhibition of TLR4-related AKT/mTOR, MAPK and NF-κB signaling pathways; in IL-4-induced M2-type macrophages, TMP down-regulated the expression of M2-related Arg1, IL-10, TGF-β, CD206 and CD163, as well as the phosphorylation levels of AKT1 and STAT6. For macrophages in a physiological state, it was very important for cells to return from a stress state to a phenotypic stability in the M0 state. These results indicated that TMP negatively regulated the M1/M2 polarization of macrophages, and made them tend to M0 homeostasis, which might provide new theoretical and data support for explaining the anti-inflammatory immunoregulatory activity of Sophora flavescens.

Immunosuppressive effects of triptolide via interleukin-2/receptor signaling

Background Triptolide (TP) has been confirmed to possess many beneficial functions including anti-inflammation and immunosuppression. Objective The present study aimed to explore the potential involvement of IL-2/IL-2R pathway in the immunosuppressive activities of TP. Methods Cultured CTLL-2 cells were utilized to evaluate the potential benefits of TP. Then cell viability was determined by CCK-8 assay, IFN-γ level by ELISA assay, Annexin V-FITC/PI double-staining and CD25 expression by flow cytometry, and protein expression by western blotting. Additionally, rhIL-2–driven lymphocytes following ConA activation were investigated. The interactions of TP with IL-2 and IL-2Rα were investigated by binding assays and molecular dynamics simulations. Results TP treatment attenuated IFN-γ level and cell viability in both rhIL-2–induced CTLL-2 cells and rhIL-2–driven splenic lymphocytes. TP treatment increased cellular apoptosis/necrosis and cleaved PARP-1 level, while suppressed c-Myc level in rhIL-2–induced CTLL-2 cells. Additionally, TP treatment reduced CD25 expression on CTLL-2 cell surface. Notably, the phosphorylation protein levels in IL-2R signaling pathways were inhibited by TP exposure prior to rhIL-2 stimulation. SPR and BLI assays verified TP that directly bound to rhIL-2 and rmIL-2Rα, respectively. Molecular simulations suggested that TP bound at the interface of IL-2 and IL-2Rα near the hydrophobic patch composed of F62, L92 on IL-2 and L23, I46, V139 on IL-2Rα, resulting in decreased binding free energy between IL-2 and IL-2Rα. Conclusions These findings collectively emphasized that TP interfered IL-2/IL-2Rα interactions, down-regulated IL-2Rα expression, and inhibited IL-2R signaling pathways activation, thereby leading to the immune cells being desensitized to rhIL-2 and exhibiting immunosuppressive properties.

12407 Assessing Human Monocyte Migration And Co-culture With Adrenocortical Cancer Cells

Abstract Disclosure: C. Hong: None. S. Feely: None. R. Challapalli: None. N. Mullen: None. A. Sorushanova: None. C. Hantel: None. M.D. Griffin: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a lipid, typically steroidogenic cancer which carries a 5-year prognosis of <10%. Therapeutic options are limited including surgery and mitotane – a poorly tolerated and efficacious insecticide. Recent data have demonstrated that the immune environment of adrenocortical carcinoma is deficient in lymphocytes while demonstrating a relative rich monocyte/macrophage population in the tumour microenvironment. We have investigated the gradient for migration of circulating peripheral monocytes to ACC. ACC cells were seeded in the bottom chamber of the transwell system. Monocytes were isolated from human peripheral blood, by negative selection using magnetic beads with high purity (>95%) and quality. Migration of monocytes at 24/48h was evaluated by identifying those remaining in the top chamber, the floating fraction in the bottom chamber and those adherent to ACC cells in the bottom chamber. Analysis of monocyte/macrophages was undertaken by flow cytometry using the following markers: Sytox Blue, CD45, CD14, CD16 and HLA-DR. At 24h, when compared to control conditions, more monocytes had migrated to the ACC cells seeded lower chamber and attached to the bottom. Similar results were observed in migration at 48h. Predominantly Classical monocytes (CD14++CD16-HLA-DR+) migrated to metastatic cancer cells (MUC1), and Non-Classical monocytes (CD14+CD16+HLA-DR+) migrated to primary cancer cells (H295R/ HAC15) at 48h. We then investigated the polarization cytokine profile of migrated monocytes with or without ACC cells at 72 h by multiplex bead-based assay. Migrated monocytes indicated a significant shift toward the M2 phenotype in the presence of H295R or HAC15. The production of the pro-inflammatory factors of IL-6, TNF-alpha, etc. decreased and immunosuppressive factors of IL-10, TARC, etc. increased. MUC1 didn’t increase M2 or M1 cytokine secretion. Moreover, surface markers of migrated monocytes were evaluated at 72h by flow cytometry. When compared to the control group, all 3 ACC cell lines switched monocytes/ macrophages into M2 phenotype, as evidenced by the decrease in the expression of the M1 marker CD86 and the increase in the expression of the M2 markers CD163 and CD206. We demonstrate monocytes migrate and associate with ACC cells: (i) subpopulations differ in transmigration properties in response to ACC cells (ii) monocytes/macrophages exhibit M2 polarization phenotype. Presentation: 6/2/2024

The human gut Bacteroides eggerthii expresses a new galactofuranose-containing lipooligosaccharide with weak immunostimulatory properties

Lipopolysaccharides (LPS) decorating the cell surface of Gram-negative bacteria exhibit nuanced functionalities linked to their precise structural composition. However, despite their critical role in health and disease, information on the structure and function of LPS from members of the human gut microbiota is still limited. Here, we deciphered the complete structure of the LPS isolated from the human gut bacterium Bacteroides eggerthii 1_2_48FAA. We showed that B. eggerthii 1_2_48FAA produces an R-type LPS (or lipooligosaccharide, LOS) composed of a heterogeneous mixture of tetra- and penta-acylated lipid A species with different degree of phosphorylation, and a compact galactofuranose-containing core oligosaccharide. Using in vitro human cell lines, we showed that B. eggerthii 1_2_48FAA LOS acts as a weak activator of TLR4-mediated signaling. Moreover, we observed that expression of maturation markers CD40, CD80 and CD86 on monocytes-derived dendritic cells upon B. eggerthii 1_2_48FAA LOS exposure was significantly lower compared to pro-inflammatory Escherichia coli LPS. Taken together, these data provide new structural and biological insights into LPS from gut bacteria, underscoring the importance of structural features in modulating host immunity.

CD151 identifies an NK cell subset that is enriched in COVID-19 patients and correlates with disease severity

Severe coronavirus disease 2019 (COVID-19) often leads to acute respiratory distress syndrome and multi-organ dysfunction, driven by a dysregulated immune response, including a cytokine storm with elevated proinflammatory cytokine levels. Natural killer (NK) cells are part of the innate immune system with a fundamental role in the defense against viral infections. However, during COVID-19 acute infection, they exhibit an altered phenotype and impaired functionality contributing to the immunopathogenesis of the disease. In this work, we have studied a cohort of patients with COVID-19 (ranging from mild to severe) by analyzing IL-15, TGF-β, PlGF and GDF-15 plasma levels and performing multiparametric flow cytometry studies. Our results revealed that severe COVID-19 patients exhibited high levels of IL-15, PlGF and GDF-15, along with an enrichment of anNK cell subset expressing the CD151 tetraspanin, which correlated with IL-15 plasma levels and disease severity. In patients, these CD151+ NK cells displayed a more activated phenotype characterized by an increased expression of HLA-DR, CD38 and granzyme B, a distinct receptor repertoire, with lower levels of CD160 and CD31 and higher levels of CD55 and, remarkably, a higher expression of tissue-resident markers CD103 and the NK cell decidual marker CD9. Last of all, in individuals with severe disease, we identified an expansion of a CD151brightCD9+ NK cell subset, suggesting that these cells play a specific role in COVID-19. Altogether, our findings suggest that CD151+ NK cells may have a relevant role in COVID-19 immunopathogenesis.

Circulating mucosal-associated invariant T cell alterations in adults with recent-onset and long-term oral lichen planus

BackgroundMucosal-associated invariant T (MAIT) cells play key roles in many inflammatory diseases. However, their effects on the long-term course of oral lichen planus (OLP) and recent-onset OLP remain unclear. In this study, we aimed to investigate the function of MAIT cells in the different processes of OLP and to explore the immunological background of this disease.MethodsThe frequency, phenotype, cytokine secretion, and clinical relevance of MAIT cells were investigated. MAIT cells were collected from the peripheral blood of 14 adults with recent-onset OLP (7–120 days after disease onset) and 16 adults with long-term course OLP (>2 years after diagnosis) using flow cytometry and compared with 15 healthy blood donors. Statistical analyses were performed using the GraphPad Prism software.ResultsMAIT cells from adults with recent-onset OLP exhibited an activated phenotype, as indicated by an increased frequency of CD69+ (p < 0.05) and CD38+MAIT cells (p < 0.01) and elevated production of the proinflammatory cytokine IL-17 A (p < 0.01), compared with healthy adult donors. In adults with long-term OLP, MAIT cells exhibited an activated and exhausted phenotype, characterized by high expression of CD69 (p < 0.01) and PD-1 (p < 0.001) and increased production of granzyme B released (p < 0.01). Compared with recent-onset OLP patients, long-term OLP patients showed a decreased production of CD8+, and CD4−CD8− cells, but an increase in PD-1+ production (p < 0.05).ConclusionsCirculating MAIT cells exhibited activation in OLP patients across varying disease durations. Given that PD-1 expression is elevated in adults with long-term OLP, it is reasonable to infer that circulating MAIT cells in long-term OLP may exhibit a more exhausted state than those in recent-onset OLP.

Recombinant ISRAA ameliorates imiquimod-induced psoriasis and knocking out Israa delays its onset.

Psoriasis, an inflammatory disease, is largely mediated by T-helper 17 cytokines. We have previously identified the immune system-released activating agent (Israa) as a novel gene that connects the nervous and immune systems. This research aims to investigate the role of the Israa gene in psoriasis in vivo using the imiquimod-induced psoriasis model. We established the model in C57BL/6 wildtype mice, which were then treated with 200 pg/mouse, 400 pg/mouse, or 800 pg/mouse of recombinant ISRAA compared to methotrexate. Subsequently, we also induced psoriasis in Israa-knockout mice to confirm the effect of Israa. Results consistently showed improvement in psoriasis in all groups receiving recombinant ISRAA. The 200 pg/mouse dose eliminated the disease, reduced the cutaneous release of IL-17 to one-third and TNF-α to one-sixth, increased IL-10 release to over 500 pg, completely resolved parakeratosis, decreased epidermal thickness to one-half, and reduced the expression of CD4 and neutrophil elastase in the skin (all p < 0.05). Israa-knockout mice exhibited less severe psoriasis in all scoring, biochemical, and histological parameters compared to wild-type mice (p < 0.05). This study highlights Israa as a crucial molecule in psoriasis and confirms its immunomodulatory role in inflammatory diseases.

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury in mice

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success in mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns in mice (10-week-old C57BL/6 J male mice weighing 25 ± 3 g). We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. We demonstrated increased intracellular calcium effects of 4-AP through Orai1-pSTAT6 signaling, where 4-AP significantly mitigated inflammatory effects by promoting M2 macrophage differentiation in in-vitro macrophages and post-skin burn tissues. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in mice treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

The Effect of Ginkgo Biloba Extract on Oxidative Stress, Anti-Inflammatory and Antiapoptotic in Vitiligo Treated with Topical Desoxymethasone

Vitiligo is a progressive and multifactorial condition of skin, mucosa, and hair depigmentation. Loss of functional melanocytes leads to the appearance of white macules on the skin, often affecting the lips and genitalia. The impact extends to psychological stress, decreased quality of life, and risk of psychiatric morbidity. Various therapeutic strategies have been designed to inhibit the immune response in vitiligo to reduce melanocyte damage while increasing melanocyte repopulation. There is no standardized method of evaluating treatment outcomes in vitiligo patients. Herbal medicines several studies have shown that GB also has many benefits for health and healing skin diseases, one of which is used in treating vitiligo. Therefore, this study aimed to evaluate the potential of Ginkgo biloba extract as a therapeutic supplement in enhancing the positive effects of desoxymethasone on vitiligo. Using a double-blind randomized control trial clinical trial research design, 26 subjects were divided into treatment and control groups. The control group received standard vitiligo therapy with topical desoxymethasone and placebo, while the treatment group received topical desoxymethasone plus Ginkgo biloba extract. Melanin index, VASI score, MDA, TNF-α, CD8 expression, and caspase 3 were measured before and after treatment. The results showed that the administration of Ginkgo biloba extract had a significant effect in reducing MDA, TNF-α, CD8 expression, and caspase 3 levels, and improving melanin index and erythema in vitiligo patients who received topical desoxymethasone therapy. These findings demonstrate the positive potential of Ginkgo biloba extract as an adjunct to vitiligo therapy, resulting in favorable clinical and molecular impacts in patients undergoing combination treatments.

Effects of PACAP Deficiency on Immune Dysfunction and Peyer's Patch Integrity in Adult Mice.

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

Design of a Robust Flow Cytometric Approach for Phenotypical and Functional Analysis of Human Monocyte Subsets in Health and Disease.

Human monocytes can be subdivided into phenotypically and functionally different classical, intermediate and non-classical monocytes according to the cell surface expression of CD14 and CD16. A precise identification and characterisation of monocyte subsets is necessary to unravel their role in inflammatory diseases. Here, we compared three different flow cytometric strategies (A-C) and found that strategy C, which included staining against CD11b, HLA-DR, CD14 and CD16, followed by several gating steps, most reliably identified monocyte subtypes in blood samples from healthy volunteers and from patients with stable coronary heart disease (CHD) or ST-elevation myocardial infarction (STEMI). Additionally, we established a fixation and permeabilisation protocol to enable the analysis of intracellular markers. We investigated the phagocytosis of lipid nanoparticles, the uptake of 2-NBD-glucose and the intracellular levels of CD74 and HLA-DM. This revealed that classical and intermediate monocytes from patients with STEMI showed the highest uptake of 2-NBD-glucose, whereas classical and intermediate monocytes from patients with CHD took up the largest amounts of lipid nanoparticles. Interestingly, intermediate monocytes had the highest expression level of HLA-DM. Taken together, we present a robust flow cytometric approach for the identification and functional characterisation of monocyte subtypes in healthy humans and patients with diseases.

Integrative analysis of H&E and IHC identifies prognostic immune subtypes in HPV related oropharyngeal cancer

BackgroundDeep learning techniques excel at identifying tumor-infiltrating lymphocytes (TILs) and immune phenotypes in hematoxylin and eosin (H&E)-stained slides. However, their ability to elucidate detailed functional characteristics of diverse cellular phenotypes within tumor immune microenvironment (TME) is limited. We aimed to enhance our understanding of cellular composition and functional characteristics across TME regions and improve patient stratification by integrating H&E with adjacent immunohistochemistry (IHC) images.MethodsA retrospective study was conducted on patients with Human Papillomavirus-positive oropharyngeal squamous cell carcinoma (OPSCC). Using paired H&E and IHC slides for 11 proteins, a deep learning pipeline was used to quantify tumor, stroma, and TILs in the TME. Patients were classified into immune inflamed (IN), immune excluded (IE), or immune desert (ID) phenotypes. By registering the IHC and H&E slides, we integrated IHC data to capture protein expression in the corresponding tumor regions. We further stratified patients into specific immune subtypes, such as IN, with increased or reduced CD8+ cells, based on the abundance of these proteins. This characterization provided functional insight into the H&E-based subtypes.ResultsAnalysis of 88 primary tumors and 70 involved lymph node tissue images reveals an improved prognosis in patients classified as IN in primary tumors with high CD8 and low CD163 expression (p = 0.007). Multivariate Cox regression analysis confirms a significantly better prognosis for these subtypes.ConclusionsIntegrating H&E and IHC data enhances the functional characterization of immune phenotypes of the TME with biological interpretability, and improves patient stratification in HPV( + ) OPSCC.

Bothrops atrox snake venom decreased MHC-II and CD86 expression in bone marrow-derived dendritic cells

The effect of Bothrops atrox venom (BaV) on the maturation of bone marrow-derived dendritic cells (BMDCs) from mice was investigated, with a focus on selected cell markers, TAP1 expression, and the release of pro-inflammatory cytokines during this process. The objective was to evaluate BaV's impact on dendritic cell (DC) function, as DCs are pivotal in antigen presentation and responsible for initiating the immune response mediated by naïve T cells, as well as regulating the immune system. Bone marrow cells were obtained from Swiss mice, and hematopoietic precursors were differentiated into BMDCs using GM-CSF and IL-4. On the 7th day, BaV and LPS were introduced into the culture, and the cells were analyzed 24 h later. BaV's ability to stimulate BMDC maturation was assessed through the analysis of surface marker expression. The findings demonstrated that BMDCs are highly influenced by culture environment factors, such as GM-CSF and IL-4, and are sensitive to additional stimuli like LPS and BaV. Mature DCs exhibited elevated levels of critical markers for T cell activation, such as MHC-II, CD80, and CD86, displaying specific phenotypic characteristics. However, the observed reduction in MHC-II and CD86 expression following BaV exposure suggests a substantial impact on the immunological activation capacity of these cells, potentially interfering with the adaptive immune response. Furthermore, the selective release of cytokines, such as IL-6, but not TNF-α or IL-1β, indicates differentiated modulation of inflammatory responses by DCs under various stimulation conditions.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T Cells in Breast Cancer Implications for Adoptive Cell Therapy.

In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+ CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Promotion of mandibular distraction osteogenesis by parathyroid hormone via macrophage polarization induced through iNOS downregulation

ObjectiveTo investigate whether Parathyroid hormone (PTH) can promote mandibular distraction osteogenesis by regulating macrophage polarization and the underlying mechanisms of this phenomenon. MethodsForty-eight Rabbits were used to establish the mandibular distraction osteogenesis experimental model, randomly divided into 2 groups. Intermittent post-operative injections of 20 μg/kg PTH and normal saline were administered to the experimental and control groups, respectively. Regenerated new bone was examined using HE staining, osteoclast numbers were determined through tartrate-resistant acid phosphatase (TRAP) staining, and macrophage polarization markers arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) expressions were elucidated using immunohistochemistry (IHC), the mRNA expression of CD206, CD11C, Arg1 and iNOS were detected using qPCR. ResultsThe bone trabeculae in the experimental group were thicker, with a more homogeneous structure and more new osteoid than in the control group. In the area of distraction osteogenesis, the osteoclast count in the experimental group was higher than in the control group (P < 0.05). IHC results indicated differential expressions of Arg1 and iNOS in the experimental group compared to the control group (P < 0.05). Relative mRNA expressions of CD11c and iNOS were lower in the experimental group than in the control group (P < 0.05), whereas the expressions of CD206 and Arg1 mRNA were higher in the experimental group compared to the control group (P < 0.05). ConclusionIntermittent PTH injections increased macrophage quantity in the mandible generated by distraction osteogenesis, downregulated iNOS, upregulated Arg1, and promoted macrophage polarization from M1 to M2 phenotype, thereby promoting mandibular distraction osteogenesis.